The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events of actomyosin motility at the molecular level. Novel methods will be applied to striated muscle fibers and to isolated motor proteins to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. The orientation of fluorescent molecules covalently bound to subunits within the myosin heads will be monitored at high time resolution by novel multi-molecule and single-molecule fluorescence polarization techniques to determine the rates and identity of specific protein structural changes. Newly developed methods of orienting spectroscopic probe molecules relative to the protein, using bifunctional attachment to engineered protein residues will be used to determine the motions of the myosin head with high time and angular resolution. An infrared optical trap will be combined with single-molecule fluorescence polarization by total internal reflection microscopy to directly evaluate the influence of mechanical stress and strain on protein orientation changes that relate to chemomechanical transduction. The experiments will be carried out on single fibers of rabbit psoas muscle and on myosins isolated from muscle, neural tissue and heterologous expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of striated muscle, neuronal development and other types of cell motility.